Cell penetrating peptides for intracellular delivery of molecules

ABSTRACT

A cell-penetrating peptide characterized in that it comprises an amino acid sequence X3X4X1X2X5X4X1X2X6X7X1X8X9X10X11X13 (SEQ ID No: 1 1), wherein X1 is F or W, X2 is F, W or Y, X3 is beta-A or S, X4 is K, R or L, X5 is E, R or S, X6 is R. T or S, X7 is E, R or S, X8 is none, F or W, X9 is P or R, X10 is R or L, X11 is K, W or R, X12 is R or F and X13 is R or K.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/428,864, filed on Feb. 9, 2017, which is a Continuation of U.S.patent application Ser. No. 14/433,570, filed on Apr. 3, 2015, now U.S.Pat. No. 9,579,395, issued on Feb. 28, 2017, which is the National Stagefiling of PCT/EP2013/070676, entitled “CELL PENETRATING PEPTIDES FORINTRACELLULAR DELIVERY OF MOLECULES” with the International Filing Dateof Oct. 4, 2013, which claims the benefit of priority fromPCT/IB2012/055343, filed on Oct. 4, 2012, each of which is herebyincorporated by reference in its entirety for all purposes as if putforth in full below.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 737372000502SEQLIST.TXT,date recorded: Oct. 25, 2018, size: 29 KB).

FIELD OF THE INVENTION

The present invention pertains to the field of intracellular delivery ofmolecules such as nucleic acids and small hydrophobic molecules. Inparticular, the invention relates to a new cell-penetrating peptide(CPP) family, which exhibits high efficacy, low toxicity and a naturaltropism for brain and lymphe node tissues.

BACKGROUND OF THE INVENTION

Although small molecules remain the major drugs used in clinic, innumerous cases, their therapeutic impact has reached limitations such asinsufficient capability to reach targets, lack of specificity,requirement for high doses leading to toxicity and major side effects.Over the past ten years, in order to circumvent limitations of smallmolecules and of gene-based therapies, we have witnessed a dramaticacceleration in the discovery of larger therapeutic molecules such asproteins, peptides and nucleic acids which present a high specificityfor their target but do not follow Lipinski's rules. Pharmaceuticalpotency of these molecules remains restricted by their poor stability invivo and by their low uptake in cells. Therefore, “delivery” has becomea central piece of the therapeutic puzzle and new milestones have beenestablished to validate delivery strategies: (a) lack of toxicity, (b)efficiency at low doses in vivo, (c) easy to handle for therapeuticapplications (d) rapid endosomal release and (e) ability to reach thetarget. Although viral delivery strategies had given much hope for geneand cellular therapies, their clinical application has suffered fromside- and toxicity-effects [1,2]. Researches were mainly focused on thedevelopment of non-viral strategies, and different methods have beenproposed including lipid, polycationic nanoparticles and peptide-basedformulations, but only few of these technologies have been efficient invivo and have reached the clinic. Cell Penetrating Peptides (CPP) areone of the most promising non-viral strategies. Although definition ofCPPs is constantly evolving, they are generally described as shortpeptides of less than 30 amino acids either derived from proteins orfrom chimeric sequences. They are usually amphipathic and possess a netpositive charge [3-5]. CPPs are able to penetrate biological membranes,to trigger the movement of various biomolecules across cell membranesinto the cytoplasm and to improve their intracellular routing, therebyfacilitating interactions with the target. CPPs can be subdivided intotwo main classes, the first requiring chemical linkage with the cargoand the second involving the formation of stable, non-covalentcomplexes. CPPs from both strategies have been reported to favour thedelivery of a large panel of cargos (plasmid DNA, oligonucleotide,siRNA, PNA, protein, peptide, liposome, nanoparticle . . . ) into a widevariety of cell types and in vivo models [3-7].

Twenty years ago, the concept of protein transduction domain (PTD) wasproposed based on the observation that some proteins, mainlytranscription factors, could shuttle within cells and from one cell toanother [for review see ref 3,4]. The first observation was made in1988, by Frankel and Pabo. They showed that the transcription-transactivating (Tat) protein of HIV-1 could enter cells and translocate intothe nucleus. In 1991, the group of Prochiantz reached the sameconclusions with the Drosophila Antennapedia homeodomain anddemonstrated that this domain was internalized by neuronal cells. Theseworks were at the origin of the discovery in 1994 of the first ProteinTransduction Domain: a 16 mer-peptide derived from the third helix ofthe homeodomain of Antennapedia named Penetratin. In 1997, the group ofLebleu identified the minimal sequence of Tat required for cellularuptake and the first proofs-of-concept of the application of PTD invivo, were reported by the group of Dowdy, for the delivery of smallpeptides and large proteins. Historically, the notion of CellPenetrating Peptide (CPP) was introduced by the group of Langel, in1998, with the design of the first chimeric peptide carrier, theTransportan, which derived from the N-terminal fragment of theneuropeptide galanin, linked to mastoparan, a wasp venom peptide.Transportan has been originally reported to improve the delivery of PNAsboth in cultured cells and in vivo. In 1997, the group of Heitz andDivita proposed a new strategy involving CPP in the formation of stablebut non-covalent complexes with their cargo [7]. The strategy was firstbased on the short peptide carrier (MPG) consisting of two domains: ahydrophilic (polar) domain and a hydrophobic (apolar) domain. MPG wasdesigned for the delivery of nucleic acids [7]. The primary amphipathicpeptide Pep-1 was then proposed for non-covalent delivery of proteinsand peptides [8]. Then the groups of Wender and of Futaki demonstratedthat polyarginine sequences (Arg8) are sufficient to drive small andlarge molecules into cells and in vivo. Ever since, many CPPs derivedfrom natural or unnatural sequences have been identified and the list isconstantly increasing. Peptides have been derived from VP22 protein ofHerpes Simplex Virus, from calcitonin, from antimicrobial or toxinpeptides, from proteins involved in cell cycle regulation, as well asfrom polyproline-rich peptides [reviews 4-6].

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C show the binding of VEPEP-3a (1A), VEPEP-3c (1B),and VEPEP-3g (1C) peptides with various fluorescently labeled cargoes(as determined by fluorescence spectroscopy).

FIG. 2 shows the particle size distribution of several VEPEP-3/cargocomplexes (VEPEP-3a/peptide, VEPEP-3c/cyclic peptide, VEPEP-3e/PNA, andVEPEP-3g/Doxo) at molar ratios ranging between 1:10 and 1:30 (asdetermined by dynamic light scattering).

FIG. 3 shows the dose-response of VEPEP-3-mediated delivery of PC4Dpeptides on viral replication in HIV-infected PBMCs. VEPEP-3a, VEPEP-3c,and VEPEP-3g peptides were used for PC4D delivery.

FIG. 4 shows the dose-response of VEPEP-3a-mediated delivery of C2 andC4 peptides on G2-arrest in Hela, MCF7, HEK-2, HS-68, and U2OS cells.

FIG. 5 shows the dose-response of VEPEP-3-mediated delivery of C2 and C4peptides on proliferation in Hela, MDA-MB, and HS-68. VEPEP-3a,VEPEP-3d, and VEPEP-3g peptides were used for C2 and C4 delivery.

FIG. 6 shows the toxicity profile of VEPEP-3 particles on Hela and U2OScells (as determined by MTT assay and by cyclophilin mRNA level).VEPEP-3a, VEPEP-3b, VEPEP-3c, VEPEP-3d, VEPEP-3e, VEPEP-3f, VEPEP-3g,and VEPEP-3h peptides were complexed with either short peptide orpeptide nucleic acid (PNA) at 1:20 molar ratio.

FIG. 7 shows the dose-response of VEPEP-3-mediated delivery of a CyclinB1 antisense PNA on Cyclin B1 protein levels in cells. VEPEP-3a andVEPEP-3h peptides were used for PNA delivery.

FIG. 8 shows the dose-response of VEPEP-3-mediated delivery ofdoxorubicin, porphyrin, or taxol on cancer cell proliferation. VEPEP-3a,VEPEP-3c, and VEPEP-3g peptides were used for delivery in MCF-7 cells.

FIGS. 9A and 9B show schematics for the formation of NANOPEP particleshaving multilayer organization.

FIGS. 10A and 10B show reduction of tumor growth by administration ofNANOPEP particles containing C2 or C4 peptide in PC3 xenograft mice.FIG. 10A shows results for intratumoral administration. FIG. 10B showsresults for intravenous administration. NANO-3A/C2: VEPEP-3a/C2 core,coated with VEPEP-3a; NANO-3A/C4: VEPEP-3a/C4 core, coated withVEPEP-3a; NANO-3F/C2: VEPEP-3f/C2 core, coated with VEPEP-3f;NANO-3F/C4: VEPEP-3f/C4 core, coated with VEPEP-3f; C2S: VEPEP-3 NANOPEPparticles with scrambled C2 peptide; C4S: VEPEP-3 NANOPEP particles withscrambled C4 peptide.

FIG. 11 shows reduction of tumor growth by intravenous administration ofNANOPEP particles containing C2 or C4 peptide in SKB3-HEK2 xenograftmice. NANO-3A/C2: VEPEP-3a/C2 core, coated with VEPEP-3a; NANO-3A/C4:VEPEP-3a/C4 core, coated with VEPEP-3a; NANO-3A-PEG/C2: VEPEP-3a/C2core, coated with PEG-VEPEP-3a; NANO-3A-PEG/C4: VEPEP-3a/C4 core, coatedwith PEG-VEPEP-3a; C2S: VEPEP-3 NANOPEP particles with scrambled C2peptide; C4S: VEPEP-3 NANOPEP particles with scrambled C4 peptide.

FIG. 12 shows reduction of tumor growth by intravenous administration ofNANOPEP particles containing antisense PNA targeting Cyclin B1 inSKB3-HEK2 xenograft mice. NANO-3A/PNA 5: VEPEP-3a/PNA core (5 μg PNA),coated with VEPEP-3a; NANO-3A/PNA 10: VEPEP-3a/PNA core (10 μg PNA),coated with VEPEP-3a; NANO-3A-PEG/PNA: VEPEP-3a/PNA core, coated withPEG-VEPEP-3a; NANO-3F/PNA 5: VEPEP-3f/PNA core (5 μg PNA), coated withVEPEP-3f; NANO-3F/PNA 10: VEPEP-3f/PNA core (10 μg PNA), coated withVEPEP-3f; NANO-3F-PEG/PNA: VEPEP-3f/PNA core, coated with PEG-VEPEP-3f.

FIG. 13 shows in vivo biodistribution of fluorescently labeled peptideor siRNA delivered by intravenous administration of VEPEP-3 NANOPEPparticles (as determined by live fluorescence animal imaging).CADY/VEPEP3/siRNA: CADY/siRNA core, coated with VEPEP-3; VEPEP-3/PEP:VEPEP-3/peptide core, coated with VEPEP-3; CADY/siRNA: CADY complexedwith siRNA.

FIGS. 14A-14D show in vivo biodistribution of fluorescently labeledpeptide or protein delivered by intravenous, intrarectal, intranasal ortransdermal administration of VEPEP-3 NANOPEP particles (as determinedby live fluorescence animal imaging). FIG. 14A shows results forintravenous administration. FIG. 14B shows results for intrarectaladministration. FIG. 14C shows results for intranasal administration.FIG. 14D shows results for transdermal administration. VEPEP-3/PROT:VEPEP-3/protein core, coated with VEPEP-3; VEPEP-3/PEP: VEPEP-3/peptidecore, coated with VEPEP-3.

DETAILED DESCRIPTION

The inventors have now designed a new family of cell-penetratingpeptides for the delivery of peptides/proteins and hydrophobicmolecules, named VEPEP-3. Delivery strategies using VEPEP-3 peptides asthe outer layer of nanoparticles are referred to as NANOPEP-3.

VEPEP-3 are short primary amphipathic peptides forming stablenanoparticles with molecules such as peptide, protein, peptide-analogue,PNA and small hydrophobic molecules, hereafter designated as “SHM”.VEPEP-3 vectors comprise the following amino acid sequence:X₃X₄X₁X₂X₅X₄X₁X₂X₆X₇X₁X₈X₉X₁₀X₁₁X₁₂X₁₃ (SEQ ID No: 11), wherein:

-   -   X₁ is F or W (independently from each other);    -   X₂ is F, W or Y (independently from each other);    -   X₃ is beta-A or S;    -   X₄ is K, R or L (independently from each other);    -   X₅ is E, R or S;    -   X₆ is R, T or S;    -   X₇ is E, R or S;    -   X₈ is none, F or W;    -   X₉ is P or R;    -   X₁₀ is R or L;    -   X₁₁ is K, W or R;    -   X₁₂ is R or F; and    -   X₁₃ is R or K.

According to a particular embodiment, this cell-penetrating peptidecomprises an amino acid sequence X₃X₁WX₂EX₁WX₂X₄X₅X₆PRX₁RX₁ (SEQ ID No:12), wherein:

-   -   X₁ is R or K (independently from each other);    -   X₂ is F, W or Y (independently from each other);    -   X₃ is beta-A or S;    -   X₄ is T or R;    -   X₅ is E or R; and    -   X₆ is W or F.

Non-limitative examples of cell-penetrating peptides according to theabove paragraph comprise an amino acid sequence selected from the groupconsisting of:

(SEQ ID No: 1) X₁KWFERWFREWPRKRR (SEQ ID No: 2) X₁KWWERWWREWPRKRK (SEQID No: 3) X₁RWWEKWWTRWPRKRK, and (SEQ ID No: 4) X₁RWYEKWYTEFPRRRR,

-   -   wherein X₁ is beta-A or S.

According to another particular embodiment of the present invention, thecell-penetrating peptide comprises the following amino acid sequence:X₃X₄X₁WX₂X₅X₁WX₂X₂WX₁X₆X₇WX₈R (SEQ ID No: 13), wherein

-   -   X₁ is F or W (independently from each other);    -   X₂ is R or S (independently from each other);    -   X₃ is beta-A or S;    -   X₄ is K, R or L;    -   X₅ is L or R;    -   X₆ is R or P;    -   X₇ is L or R; and    -   X₈ is R or F.

According to a particular embodiment of the above cell-penetratingpeptide, X₈ is R.

Non-limitative examples of cell-penetrating peptides according to theabove paragraphs comprise an amino acid sequence selected from the groupconsisting of:

(SEQ ID No: 5) X₁RWWRLWWRSWFRLWRR (SEQ ID No: 6) X₁LWWRRWWSRWWPRWRR (SEQID No: 7) X₁LWWSRWWRSWFRLWFR, and (SEQ ID No: 8) X₁KFWSRFWRSWFRLWRR,

-   -   wherein X₁ is beta-A or S.

The present invention also pertains to a stapled cell-penetratingpeptide derived from a VEPEP-3 cell-penetrating peptide as describedabove. A “stapled” peptide designates a peptide which comprises achemical linkage (in addition to the amino acid chain) between tworesidues. In a particular embodiment of stapled VEPEP-3 peptides, theVEPEP-3 peptide comprises a hydrocarbon linkage between two residueswhich are separated by three or six residues. The skilled artisan canobtain these peptides by using techniques which are available in theart, for example as described by Verdine and Hilinski, Methods inEnzymology, 2012 [12].

A particular embodiment of stapled VEPEP-3 according to the presentinvention comprises an amino acid sequence derived from SEQ ID No: 12 byaddition of a non-natural amino acid between the amino acids inpositions 2 and 3 of said sequence, replacement of the amino acid inposition 9 of SEQ ID No: 12 by a non-natural amino acid, and addition ofa hydrocarbon linkage between these two non-natural amino acids. Anexample of such a stapled VEPEP-3 CPP comprises the amino acid sequenceX₁KX₂WWERWWRX₃WPRKRK (SEQ ID No: 9), wherein X₁ is a beta-alanine or aserine and wherein X₂ and X₃ are non-natural amino acids used for thebinding of a hydrocarbon linkage.

Another embodiment of stapled VEPEP-3 according to the present inventioncomprises an amino acid sequence designed by replacement of the aminoacids in position 5 and 12 of SEQ ID No: 13 by non-natural amino acids,and addition of a hydrocarbon linkage between the two non-natural aminoacids (it being understood that the synthesis process directlyintegrates the non-natural amino acids). For example, a stapled VEPEP-3peptide comprises the amino acid sequence X₁RWWX₂LWWRSWX₃RLWRR (SEQ IDNo: 10), wherein X₁ is a beta-alanine or a serine, and wherein X₂ and X₃are non-natural amino acids used for the binding of a hydrocarbonlinkage.

VEPEP-3 strategy improves both ex-vivo and in vivo delivery andefficiency of peptide/protein/peptide analogue and small hydrophobicmolecules, without activating the innate immune response or inducingtoxic side effects.

According to a preferred embodiment, a cell-penetrating peptide of thepresent invention further comprises, covalently linked to the N-terminalend of the amino acid sequence, one or several chemical entitiesselected in the group consisting of an acetyl, a fatty acid, acholesterol, a poly-ethylene glycol, a nuclear localization signal, anuclear export signal, an antibody, a polysaccharide and a targetingmolecule (peptide, fatty acid, saccharide).

As developed below and shown at least in example 5 below, PEGylation ofVEPEP-3 peptides is particularly advantageous for stabilizingnanoparticles in vivo.

In addition or alternatively, a cell-penetrating peptide according tothe invention can comprise, covalently linked to the C-terminal end ofits amino acid sequence, one or several groups selected in the groupconsisting of a cysteamide, a cysteine, a thiol, an amide, anitrilotriacetic acid optionally substituted, a carboxyl, a linear orramified C1-C6 alkyl optionally substituted, a primary or secondaryamine, an osidic derivative, a lipid, a phospholipid, a fatty acid, acholesterol, a poly-ethylene glycol, a nuclear localization signal,nuclear export signal, an antibody, a polysaccharide and a targetingmolecule.

Another aspect of the present invention is a complex comprising acell-penetrating peptide as described above and a cargo selected amongstprotein/peptide and hydrophobic molecules. Examples of polypeptidecargoes are small peptide or protein, cyclic peptide, peptide-basedbiomarker, bio-drug, PNA or uncharged oligonucleotides. In a preferredembodiment of the complex according to the invention, the cargo is asmall molecule (size lower than 1.5 kDa), either hydrophobic or charged.Preferred cargos in the complexes according to the present invention areanticancer and antiviral drugs. Non-limitative examples of smallhydrophobic molecules which can be used include amino acids, di- ortri-peptides (labelled or not) daunomycin, Paclitaxel, doxorubicin, AZT,porphyrin, fluorescently-labelled-nucleosides or nucleotides(FAM-Guanosine, CY5_UTP, CY3-UTP), hydrophobic maghemite (contrastagents or magnetic nanoparticles Fe₂ O₃) and fluorescent dyes.

The size of the complexes described above is preferably between 50 and300 nm, more preferably between 50 and 200 nm (the size of the complexherein designates its mean diameter).

In the complexes according to the invention, the cargo/VEPEP-3 molarratio depends on the nature and size of the cargo, but is generallycomprised between 1/1 and 1/50. For small peptide cargoes, thecargo/VEPEP-3 molar ratio preferably ranges from 1/5 to 1/20. For smallmolecule cargoes, the cargo/VEPEP-3 molar ratio preferably ranges from1/3 to 1/10. For large protein cargoes, the cargo/VEPEP-3 molar ratiopreferably ranges from 1/10 to 1/40.

According to an advantageous embodiment of the complexes as describedabove, the VEPEP-3 peptides comprise a polyethylene glycol group or anacetyl group covalently linked to their N-terminus, and/or a cysteamidegroup covalently linked to their C-terminus.

The above complexes can be advantageously used as “core shells” forobtaining bigger complexes, or nanoparticles, by an additional step ofcoating the cargo/VEPEP-3 complex with another layer of cell-penetratingpeptides, which can be different from the VEPEP-3 peptides describedabove. Examples of such nanoparticles are VEPEP-3/CADY (wherein CADY isa CPP as described in EP1795539 and in [11], for example CADY-1:Ac-GLWRALWRLLRSLWRLLWKA-cysteamide (SEQ ID No: 28)), VEPEP-3/PEP-1(wherein Pep-1 is a CPP as described in [8]), VEPEP-3/MPG (wherein MPGis a CPP as described in U.S. Pat. No. 7,514,530 and in [7, 10]), aswell as nanoparticles with an outer layer made of a CPP belonging toanother VEPEP family, for example selected from the following list:

VEPEP-6a: (SEQ ID No: 29) Ac-X₁LFRALWRLLRSLWRLLWK-cysteamide VEPEP-6b:(SEQ ID No: 30) Ac-X₁LWRALWRLWRSLWRLLWKA-cysteamide VEPEP-6c: (SEQ IDNo: 31) Ac-X₁LWRALWRLLRSLWRLWRKA-cysteamide VEPEP-6d: (SEQ ID No: 32)Ac-X₁LWRALWRLWRSLWRLWRKA-cysteamide VEPEP-6e: (SEQ ID No: 33)Ac-X₁LWRALWRLLRALWRLLWKA-cysteamide VEPEP-6f: (SEQ ID No: 34)Ac-X₁LWRALWRLLRNLWRLLWKA-cysteamide EPEP-9a1: (SEQ ID No: 35)Ac-X₁LRWWLRWASRWFSRWAWWR-cysteamide VEPEP-9a2: (SEQ ID No: 36)Ac-X₁LRWWLRWASRWASRWAWFR-cysteamide VEPEP-9b1: (SEQ ID No: 37)Ac-X₁RWWLRWASRWALSWRWWR-cysteamide VEPEP-9b2: (SEQ ID No: 38)Ac-X₁RWWLRWASRWFLSWRWWR-cysteamide VEPEP-9c1: (SEQ ID No: 39)Ac-X₁RWWLRWAPRWFPSWRWWR-cysteamide VEPEP-9c2: (SEQ ID No: 40)Ac-X₁RWWLRWASRWAPSWRWWR-cysteamide VEPEP-9d: (SEQ ID No: 41)Ac-X₁WWRWWASWARSWWR-cysteamide VEPEP-9e: (SEQ ID No: 42)Ac-X₁WWGSWATPRRRWWR-cysteamide VEPEP-9f: (SEQ ID No: 43)Ac-X₁WWRWWAPWARSWWR-cysteamide ST-VEPEP-6a: (SEQ ID No: 44)Ac-X₁LFRALWR_(s)LLRS_(s)LWRLLWK-cysteamide ST-VEPEP-6aa: (SEQ ID No: 45)Ac-X₁LFLARWR_(s)LLRS_(s)LWRLLWK-cysteamide ST-VEPEP-6ab: (SEQ ID No: 46)Ac-X₁LFRALWS_(s)LLRS_(s)LWRLLWK-cysteamide ST-VEPEP-6ad: (SEQ ID No: 47)Ac-X₁LFLARWS_(s)LLRS_(s)LWRLLWK-cysteamide ST-VEPEP-6b: (SEQ ID No: 48)Ac-X₁LFRALWRLLR_(s)SLWS_(s)LLWK-cysteamide ST-VEPEP-6ba: (SEQ ID No: 49)Ac-X₁LFLARWRLLR_(s)SLWS_(s)LLWK-cysteamide ST-VEPEP-6bb: (SEQ ID No: 50)Ac-X₁LFRALWRLLS_(s)SLWS_(s)LLWK-cysteamide ST-VEPEP-6bd: (SEQ ID No: 51)Ac-X₁LFLARWRLLS_(s)SLWS_(s)LLWK-cysteamide ST-VEPEP-6c: (SEQ ID No: 52)Ac-X₁LFAR_(s)LWRLLRS_(s)LWRLLWK-cysteamide,as well as variants thereof (regarding the amino acid sequence and/orthe N- and C-terminal chemical groups), wherein X₁ is beta-A or S andwherein the residues followed by an inferior “s” are linked by ahydrocarbon linkage. Preferred variants of the above sequences forforming nanoparticles according to the invention are PEGylated at theirN-terminal extremity instead of being acetylated.

Another aspect of the present invention pertains to nanoparticles madeof a “core shell” comprising a cargo and a first carrier molecule,surrounded by VEPEP-3 peptides. These are herein referred to as“NANOPEP-3” particles. NANOPEP-3 technology constitutes a “custom-built”delivery system containing a common core particle, trapping therapeuticmolecule, with surface VEPEP-3 peptides which are preferablyfunctionalized for tumour or tissue targeting in vivo. From a structuralpoint of view, NANOPEP-3 particles are constituted by a “core” which iscoated by a layer of VEPEP-3 peptides. The “core” corresponds to acomplex comprising a cargo and a vector or carrier such as a firstcell-penetrating peptide, a liposome, a polycationic structure, a carbonnanoparticle, etc. In NANOPEP-3 particles, the layer of VEPEP-3 peptides(peripheral peptide) stabilizes the particle and can be functionalized.Functionalizing NANOPEP-3 particle surface with either cholesterol,lipid, PEG-molecules improves particles stability in vivo, favours theiradministration by either systemic or topical route and allows rapidliberation of active cargoes within tumor cells or tissues.Functionalization of the surface of NANOPEP-3 particles with small FABfragments, peptides, antibodies and lipids has been shown to favour invivo tissue or tumor targeting. Also, Functionalizing NANOPEP-3 particlesurface with polysaccharide such as PLGA, can be used as formulation forslow release of drug and cargo and allow a long term response in vivo.As shown in Example 5 below, the inventors have observed that N-terminalPEGylation of at least part of the VEPEP-3 peptides surrounding theNANOPEP-3 particles increases the biodistribution of cargoes in thetumour, probably by stabilizing the NANOPEP-3 particles in the plasma.

NANOPEP-3 technology improves both cellular and in vivo delivery ofbiologically active cargoes and has been validated on a large set ofcell lines including adherent and suspension cell lines, hard totransfect cell lines. NANOPEP-3 particles strongly interact with cellmembranes and enter the cell independently of the endosomal pathway orrapidly escape from early endosomes. NANOPEP-3 technology presentsseveral advantages including rapid delivery with very high efficiency,stability in physiological buffers, protection of the cargo againstdegradation, lack of toxicity and of sensitivity to serum, ability offorming mix nanoparticles, can be functionalized and have beensuccessfully applied to the delivery of different types of cargoes intoa large variety of cell lines as well as in animal models, therebyconstituting powerful tools for basic research and therapeuticapplications. NANOPEP-3 technology can be applied both at therapeuticand diagnostic/theragnostic levels, as well as for imaging, for examplebrain imaging.

In a particular embodiment of NANOPEP-3 particles according to thepresent invention, the cargo is complexed to a first cell-penetratingpeptide, which can be, for example, selected amongst CADY, MPG, PEP-1,PPTG1, poly Arginine motif, VEPEP-family peptide (VEPEP-3, VEPEP-6,VEPEP-9, stapled or not) as described above (such as SEQ ID Nos: 1 to 13and 19 to 52 and variants thereof), or any other known CPP. Thiscargo/CPP complex is then coated with a layer of VEPEP-3 peptides.According to this embodiment, the skilled artisan will advantageouslychoose the first CPP depending on the nature of the cargo, so that thecomplex of cargo and first CPP is stable. Hence, a wide diversity ofcargoes can be included in NANOPEP-3 particles.

In the nanoparticles as above-described, the core/VEPEP-3 molar ratiodepends on the nature and size of the core, but is generally comprisedbetween 1/1 and 1/50. For small peptide/CPP cores, the core/peripheralVEPEP-3 molar ratio preferably ranges from 1/5 to 1/30, depending on thenature of peptide cargo (hydrophobicity and charge).

In a preferred embodiment of the nanoparticles according to theinvention, the size of the nanoparticle is between 20 and 300 nm.

According to an advantageous embodiment of the NANOPEP-3 particlesaccording to the invention, the VEPEP-3 peptides forming the peripherallayer of the nanoparticles comprise a poly-ethylene glycol or an acetylgroup covalently linked to their N-terminus, and/or a cysteamide groupcovalently linked to their C-terminus.

According to another preferred embodiment, the core shell of theparticles is coated with a VEPEP-3 peptide functionalized with NTA (forexample, a VEPEP-3 peptide with nitrilotriacetic acid covalently linkedto its C-terminus). This allows the subsequent attachment to the surfaceof the particle, of any protein (or other molecule) harboring ahistidine tag. This strategy offers the major advantage of having acommon two-layers particles “NANOPEPHIS-3” which can be associated toany His-tagged molecule.

In particular embodiments of the complexes and nanoparticles accordingto the invention, at least part of the VEPEP-3 cell-penetrating peptidesare bound to a targeting molecule. In the case of NANOPEP-3 particles,at least part of the cell-penetrating peptides which are at theperiphery of the nanoparticle are preferentially bound to a targetingmolecule. Examples of targeting molecules include antibodies, nanobodiesand Fc or FAB fragments (for example targeting HEK2/MUC1/EGF/XCCR4),ligands, especially targeting receptors which are over-expressed at thesurface of certain cell-types and homing peptides specific of selectedorgans. Non-limitative examples of such ligands and homing peptides are:RGD-peptide, homing targeting peptides (brain NT1 peptide, Ganglion GM1peptide, as well as all other previously described peptides for tissuesand cell line targeting), folic acid, polysaccharides, and matrixmetalloprotease targeting peptide motif (MMP-9 or MMP3 for tumourselectivity).

According to a particular embodiment of the present invention, thecomplexes or nanoparticles are formulated se that they can be storedduring several months without losing their stability and functionalefficacy. As disclosed in example 5 below, the complexes andnanoparticles of the invention can advantageously be lyophilized in thepresence of a sugar. Non-limitative examples of sugars which can be usedto that aim are sucrose, glucose, manitol and a mix thereof, and theycan be used, for example, in a concentration ranging from 5% to 20%,preferably 5% to 10%, it being understood that a concentration of 5% isobtained by adding 5 grams per litre of solution before lyophilization.

Another aspect of the present invention is the use of a complex ornanoparticle as above-described, as a medicament and as a marker or animaging agent.

In particular, the VEPEP-3/cargo complexes and NANOPEP-3 particles canadvantageously be used in the treatment of a brain disease and/or of alymph node disease, for example by targeting a latency pathogenlocalized in the brain and/or in a lymph node. They can also be used forbrain and/or lymph node imaging.

The present invention also pertains to a therapeutic, cosmetic ordiagnostic composition comprising a complex or a nanoparticle asdescribed above. For example, a composition comprising a complex ornanoparticle having a peptide targeting protein/protein interactions,involving essential protein CDK and Cyclin required for cell cycleprogression as a cargo, and a targeting molecule specific for tumourcells (for example: RGD-peptide, folic acid, MUC-1 or HEK2 antibodies ornanobodies), is part of the present invention. Depending on theapplication, this composition can be formulated for intravenous,intratumoral, topical, intrarectal, intranasal, transdermal, orintradermal administration, or for administration via a mouth spray, orfor administration as a subcutaneous implant for slow release of a drug.

The present invention also pertains to a method for delivering amolecule into a cell in vitro, comprising a step of putting said cellinto contact with a complex or nanoparticle as described above.

Several aspects of the present invention are further developed in thefollowing examples, illustrated by the figures (which are described inthe examples).

Example 1: Materials and Methods VEPEP-3 Peptides

All peptides were synthesized by solid-phase peptide synthesis usingAEDI-expensin resin with (fluorenylmethoxy)-carbonyl (Fmoc) on a PioneerPeptide Synthesizer (Pioneer™, Applied Biosystems, Foster City, Calif.)starting from Fmoc-PAL-PEG-PS resin at a 0.2 mmol scale. The couplingreactions were performed with 0.5 M of (HATU in the presence of 1 M ofDIEA. Protecting group removal and final cleavage from the resin werecarried out with TFA/Phenol/H₂O/Thioanisol/Ethanedithiol(82.5/5/5/5/2.5%) for 3 h30 min. All the peptides presented a cysteamidegroup at the C-terminus and were acetylated at the N-terminus. Thepeptide synthesis started by the C-terminus, using an AEDI-expensinresin starting with a cysteamide link, as described by Mery et al., 1992[9]. All the peptides contained a beta-Alanine or a serine at theN-terminus to favour any further functionalization without using theC-terminal cysteamide group.

Functionalization of Vepep-3

Two approaches were used for peptide functionalization

-   -   (1) Peptide conjugations with peptide, antibody, pegylation,        NTA, cholesterol, stearylation, were performed at the primary        amino group of the N-terminal residue, through a beta alanine or        serine. It is advantageous to maintain the C-terminal cysteamide        free, since it is known to be required to stabilize the particle        through disulfide bounds (SH-SH). Functionalized peptides were        further purified by Reverse Phase-HPLC and analyzed by        electro-spray ionization mass spectroscopy.    -   (2) Peptide conjugations were also performed via disulfide bound        using the SH-group of the cysteamide moiety of the peptide.

VEPEP-3-Funct-1: (SEQ ID No: 14) X-LWWRRWWSRWWPRWRR-CH₂—CH₂—SHVEPEP-3-Funct-2: (SEQ ID No: 15) Ac-LWWRRWWSRWWPRWRR-CH₂—CH₂—S—S-XVEPEP-3-Funct-3: (SEQ ID No: 16) X-W(W-F)RLW(W-F)RLR-CH₂—CH₂—SHVEPEP-3-Funct-4: (SEQ ID No: 17) Ac-W(W-F)RLW(W-F)RLR-CH₂—CH₂S—S-X

-   -   X: Cholesterol, Pegylation, stearyl, palmitoyl, small FC or FAB        fragments, nanobody, nitrilotriacetic acid (2×NTA),        tissue-targeting peptides (brain, lung, lymph node, pancreas . .        . ).

VEPEP-3 Structure

VEPEP-3 peptides are primary amphipathic peptides; they are highlyversatile and show a strong structural polymorphism. VEPEP-3 peptidesare unfolded in solution as a free form and adopt an alpha helicalconformation in the N-terminal part in the presence of lipid orartificial cellular membranes as well as in the presence of cargos suchas peptide or protein.

Peptides and Proteins

Peptides targeting CDK/Cyclin (C4, C2 sequences of SEQ ID Nos: 20 to 23)or HIV integrase (PC4 & PC6 sequences of SEQ ID Nos: 24 to 27) linear orcyclic version were obtained for Polypeptide.

(SEQ ID No: 20) C2: KKQVLAMEHLVT (SEQ ID No: 21) C2S: VTLMEAKKQVLT (SEQID No: 22) C4: KKQVRMAHLVLT (SEQ ID No: 23) C4C: CKKQVRMAHLVLTC (SEQ IDNo: 24) PC4, also noted PC4D RWTEWEWW (SEQ ID No: 25) PC4S: TWFTEWFT(SEQ ID No: 26) PC6, also noted PC6D: KWETWWET (SEQ ID No: 27) PC6S:KAETWAET

-   -   Proteins; including GFP overexpressed in E. coli and short        protein nanobodies, corresponding to chamelidea antibodies were        also expressed in E. coli.

Oligonucleotides & PNA

Short oligonucleotides, PNA and 5′ Alexa⁷⁰⁰ or Cy5 fluorescentlylabelled PNA were synthesized by Eurogentec (Belgium) according to thefollowing sequences:

(SEQ ID No: 18) Cyc-B1a; TGC CAT CGG GCT TGG AGG-^(CY5) (SEQ ID No: 19)Cyc-Bct; TGC CAT CAA GCT TAG AGG-^(CY5)

Fluorescence Titrations

Fluorescence experiments were performed on a PTI spectrofluorimeter at25° C. in a NaCl 154 mM buffer. Intrinsic Trp-fluorescence of VEPEP-3was excited at 290 nm and emission spectrum was recorded between 310 and400 nm, with a spectral band-pass of 2 and 8 nm for excitation andemission, respectively. FITC-fluorescence of labelled-peptide wasexcited at 492 nm and emission recorded between 500 and 580 nm. ForVEPEP-3/peptide interaction, 0.5_(R)M of FITC-labelled peptide wastitrated by increasing concentrations of VEPEP-3. All measurements werecorrected for the dilution and curve fitting were performed by usingGrafit software (Erithacus).

Characterization of Peptide-Based Nanoparticles

Mean particle size distribution was determined with a Coulter N4 Plus(Coulter-Beckman) at 25° C. for 3 mM per measurement and zeta potentialwas measured with Zetasizer 4 apparatus (Malvern Ltd,)

Cell Culture and VEPEP-Mediated Cargo Delivery

Adherent HS68 fibroblasts, HeLa, PC3, MCF-7, SCK3-Her2, PBMC cell lines(from American Type Culture Collection (ATCC)) were cultured inDulbecco's Modified Eagle's Medium supplemented with 2 mM glutamine, 1%antibiotics (streptomycin 10,000 μg/ml, penicillin, 10,000 IU/ml) and10% (w/v) foetal calf serum (FCS), at 37° C. in a humidified atmospherecontaining 5% CO₂. Stock solutions of VEPEP-3/peptide particles wereprepared by complexing 1 μM peptide with VEPEP-3 peptides at a molarratio of 1/20 for 30 min at 37° C. Lower concentrations ofVEPEP-3-carrier/peptide (from 500 nM to 1 μM) were obtained by serialdilution of the stock complexes in PBS, in order to preserve the sameVEPEP-3-carrier/peptide ratio. 150,000 cells seeded in a 35 mm dish theday prior transfection, were grown to 60% confluence and overlaid with200 μl of preformed complexes, incubated for 3-5 min, then 400 μl ofDMEM were added. After 30 min. incubation at 37° C., 1 ml of fresh DMEMcontaining 16% foetal calf serum (FCS) was added in order to reach afinal FCS concentration of 10%, without removing the overlay ofVEPEP-3/peptide complexes. Cells were returned to the incubator for 24hrs. For cdk4 and CDK2 derived peptides cell proliferation was monitoredafter 24 and 48 hrs. For peptide-targeting integrase, HIV proliferationwas analyzed on activated PBMC cells after 3 and 5 days. Data reportedare an average of 3 or 4 distinct experiments.

Cytotoxicity

Toxicity of VEPEP-3/peptide or VEPEP-3/protein complexes wasinvestigated on Hela and HS-68 cell lines. 30,000 cells seeded in24-well plated the day prior transfection, were incubated withincreasing concentrations of peptide or protein complexed with VEPEP-3at a 20/1 molar ratio ranging from 1 to 5 μM (500 μM VEPEP-3), for 30min prior to addition of medium to reach a final 10% concentration ofFCS. Cytotoxic response was measured 12 hr or 24 hr later by monitoringthe housekeeping gene cyclophilin mRNA level (Quantigen, Panomic Inc.)and by colorimetric MTT assay (Sigma, Germany), respectively. For MTTassay, cell culture medium was removed and replaced with PBS containing2.5 mg/ml of MTT for 4 hr. Results correspond to the average of 3separate experiments.

Mouse Tumour Models

Athymic female nude mice (6-8 weeks of age) were subcutaneouslyinoculated into the flank with 1×10⁶ PC3, A549 or SCK-3-HEK2 cells in100 μl PBS. Two to three weeks after tumour implant, when tumour sizereached about 100 mm³, animals were treated by intratumoral orintravenous injection, every 3 days, with a solution of 0.1 ml of eitherfree CDK2 or CDK4 derived peptide (200 μg), control scramble peptide C4Cor C4 or C2 peptides (10, 50, 100 μg) complexed with NANOPEP-3 at a 1/20molar ratio. Tumour diameter was measured in two directions at regularintervals using a digital calliper and tumour volume was calculated aslength×width×height×0.52. Curves show the mean value of tumour size in acohort of six animals and neither animal death nor any sign of toxicitywere observed. Experiments were performed according to nationalregulations and approved by the local animal experimentation ethicalcommittee. The statistical significance of the results was calculated byStudent's t test and p<0.05 considered to be statistically significant.

In Vivo Imaging of Peptide Biodistribution

In vivo fluorescence imaging was performed as previously described byCrombez et al, 2009, Nucleic Acid Res [10]. Mice were injectedintravenously with 100 μg (200 μl) of Alexa700 fluorescently labelledpeptide (C4) either naked or complexed with VEPEP-3 (n=4 animals pergroup). Anaesthetized mice, using 2% Isoflurane, were illuminated by 663nm light emitting diodes equipped with interference filters and movieswere acquired over the first 15 minutes and fluorescence images weretaken every hour for 5 hrs and then after 24 hrs, with a back-thinnedCCD cooled camera as previously described (Crombez et al, 2009, NucleicAcid Res). At 24 hr mice were euthanized and different organs wereremoved for quantification of Alexa fluorescence.

Example 2: VEPEP-3 Peptides Applications for Molecules Delivery Example2.1: VEPEP-3 Peptides Form Stable Nanostructures with Peptides andProteins

VEPEP-3 peptide form stable complexes with peptides and proteins. Thebinding of cargos to VEPEP-3 was monitored by fluorescence spectroscopyusing the two intrinsic Trp groups of VEPEP-3 (3 to 5 Trp-residues) andextrinsic fluorescently labelled cargoes (using Cy3, Cy5 or FITC). Curvefitting reveal that VEPEP-3 strongly binds the different cargoes withdissociation constant in the nanomolar range (examples with VEPEP-3a,VEPEP-3C, and VEPEP-3g and three different cargoes are reported in FIGS.1A-1C, all the data are reported in Table 1).

Example 2.2: VEPEP-3 Peptides Form Stable Nanostructures with SmallHydrophobic Molecules

VEPEP-3 peptides also form stable particles with small aromaticmolecules including Daunomycin, Paclitaxel, doxorubicin, porphyrin andcharged molecules including nucleotide, nucleoside and peptide-analog ofnucleic acids or fluorescent dyes (FIGS. 1A-1C). The dissociationconstant for small hydrophobic molecule ranges between 0.05 to 2 μM,depending on the nature of the dyes and of the peptides.

TABLE 1 VEPEP-3/Cargo complexes characterization. Cargoes SEQ peptideCyclic peptide Protein SHM PNA ID Kd Kd Kd Kd Kd VEPEP-3 No Binding (nM)Binding (nM) Binding (μM) Binding (μM) Binding (nM) VEPEP-3a 1 yes 10-20yes 50-100 Yes 1   Yes 1 Yes 100 VEPEP-3b 2 yes 10-20 yes 50-100 Yes 0.5Yes 0.5 Yes 200 VEPEP-3c 3 yes 10-20 yes 50-100 Yes 0.2 Yes 0.2 Yes 200VEPEP-3d 4 yes 10-20 yes 50-100 Yes 0.1 Yes 0.1 Yes 100 VEPEP-3e 5 yes10-20 yes 5-20 Yes 0.02-0.1 Yes 0.02 Yes 5-50 VEPEP-3f 6 yes 10-20 yes5-20 Yes 0.02-0.1 Yes 0.02 Yes 5-50 VEPEP-3g 7 yes 10-20 yes 5-20 Yes0.02-0.1 Yes 0.02 Yes 5-50 Peptide (C4 and C2), Cyclic peptide (PC4),protein (GFP & Nanobody), SHM: Doxorubicine small hydrophobic moleculeand PNA (15 mer-PNA).

Example 2.3: VEPEP-3 Peptides Form Stable Nanoparticles with theirDifferent Cargoes

The size of the particles was monitored by dynamic light scattering. Theoptimal VEPEP-3 peptide/cargo molar ratio is ranging between 1/10 to1/30, FIG. 2 reports DLS analysis of VEPEP-3a/peptide, VEPEP3c/cyclicpeptide, VEPEP-3e/PNA and VEPEP-3g/Doxo particles formed at ratio 1/20(FIG. 2). The size of the particles is of about 100 to 200 nanometers indiameter.

Example 3: VEPEP-3 APPLICATIONS in CULTURED CELLS Example 3.1: VEPEP-3Mediated Delivery of Peptide and Cyclic Peptide in Different Cell Lines

VEPEP-3 peptides have been used for the delivery of different peptidesinto different cell lines, including primary cell lines, stem cell linesand challenging cell lines. Peptide delivery was monitored using threeapproaches: fluorescence spectroscopy and monitoring of biologicalresponses (anti proliferation and anti viral responses)

-   -   1—Fluorescent labelled peptide was visualized in the different        cell lines using fluorescence microscopy or FACS sorting (Table        2). In most of the cell lines, the uptake of Cy-5 labelled        peptides is more than 70% of the cells.    -   2—Dose-response experiments performed on different cultured        cells revealed that VEPEP-3-mediated delivery of C2 and C4        peptides, targeting either cdk2/cyclin A or CDK4/cyclin D        complexes, blocks cell proliferation of different cancer cells.    -   3—Dose-response experiments performed on HIV infected activated        PBMC cells revealed that VEPEP-3-mediated delivery of PC4D        peptides, targeting pre-integration complex and HIV integrase,        blocks viral replication (the effect of PC4D in complex with        VEPEP-3a, VEPEP-3c and VEPEP-3g is shown on FIG. 3).

TABLE 2 Efficiency FACS Cell lines origin analysis of Cy-5 C4 Hela Humanepithelial cervical cancer cells 75% Jurkat Human T lymphocyte 90% HepG2Human hepatocyte 70% C2C12 Mouse myoblast 90% MEF Mouse fibroblast 90%HS-68 Human fibroblast 90% CEM-SS Human macrophage 80% U2OS Humanosteoblast 91% MCF7 Human breast adenocarcinoma 70% MT4 Human Tlymphocyte 70% HER2 Human breast cancer 90% MDA-MB Human breast cancer70% PBMC Human macrophage 90%

Example 3.2: VEPEP3-Mediated Delivery of Peptide Targeting Cdk2/Cyclin Aor CDK4/Cyclin D Induces G2 Arrest & Blocks Cancer Cell Proliferation

Dose-response experiments performed on cultured cells revealed thatVEPEP-3 mediated delivery of C2 and C4 peptide induced a robustbiological response associated with specific cell cycle arrest in G2(FIG. 4). A peptide C2 or C4 concentration of 200 nM was sufficient toblock proliferation of Hela, MCF7, HEK-2, HS-68 and U2OS cells. IC₅₀ of35±12 nM and 37±15 nM were estimated for C4 and C2 peptides,respectively on Hela, of 58±6 nM and 67±15 for C4 and C2 peptides,respectively on MCF7, and of 78±10 nM and 102±24 nM for C4 and C2peptides, respectively, on HEK-2. In contrast, proliferation was onlyreduced by 10 to 20% for non-transformed HS68 fibroblasts, in perfectagreement with the impact of the check point G2-M on the cell cycleproliferation and showing the specificity of the peptide for cancercells. FIG. 5 shows an example with VEPEP-3a, VEPEP-3d and VEPEP-3g.

C2 and C4 mediated dissociation of CDK2/cyclin A or CDK4/cyclin Dcomplex was directly associated with accumulation of cells with a 4Ncontent, consistent with downregulation of Cdk1-Cyclin B1 activity, andwas optimally obtained with 200 nM peptide and IC₅₀ values estimated to36±21 nM and 46±14 nM for HeLa and MDA_MB cells, respectively (FIG. 5).FIG. 4 shows an example using VEPEP-3a with C2 and C4 at molar ratio1/20. In contrast, no effect on cell cycle progression was observed with500 nM of a scrambled C2 peptides complexed with VEPEP-3a at a 20/1ratio, or with VEPEP-3a carrier alone (200 μM).

Example 3.3: VEPEP3-Mediated Delivery of Peptide Targeting HIV IntegraseBlocks HIV Virus Replication

The anti-HIV activities of the peptides (PC4D, PC6D & PC4S) andVEPEP-3/peptides were assayed according to previously described method(Roisin et al, 2004). Phytohemagglutinin-P (PHA-P)-activated peripheralblood mononuclear cells (PBMC) treated by increasing concentrations ofpeptide (from 100 to 0.1 nM), one hour later, were infected with hundred50% tissue culture infectious doses (TCID₅₀) per 100,000 cells of theHIV-1-LAI or different resistant strains (Barre-Sinoussi et al, 1983).Viruses were amplified in vitro on PHA-P-activated PBMC. Viral stock wastitrated using PHA-P-activated PBMC, and 50% TCID₅₀ were calculatedusing Karber's formula (Karber 1931). Samples were maintained throughoutthe culture, and cell supernatants were collected at day 7post-infection and stored at −20 C. Viral replication was measured byquantifying RT activity in cell culture supernatants. In parallel,cytotoxicity of the compounds was evaluated in uninfectedPHA-P-activated PBMC by colorimetric 3-(4-5 dimethylthiazol-2-yl)2,5diphenyl tetrazolium bromite (MTT) assay on day 7 (Mossmann 1983).Experiments were performed in triplicate and repeated with another blooddonor. Data analyses were performed using SoftMax®Pro 4.6 microcomputersoftware: percent of inhibition of RT activity or of cell viability wereplotted vs. concentration and fitted with quadratic curves; 50%effective doses (ED₅₀) and cytotoxic doses (CD₅₀) were calculated.

Dose-response experiments performed on cultured cells revealed thatVEPEP-3a, VEPEP-3c and VEPEP-3g mediated delivery of PC4D and PC6Dsignificantly blocks viral replication on PBMC infected by HIV-1_(LA)).(FIG. 3). When associated to the carrier peptide VEPEP-3, PC4D and PC6Dexhibit a 15-fold higher antiviral activity than AZT, which is thereference RT-inhibitor currently used in clinic: IC₅₀ of 0.12±0.05 nMand 0.09±0.05 nM, respectively. In contrast, the scrambled peptides PC4Sand PC6S do not show any anti viral activity. Both VEPEP-3/peptidecomplexes do not induce a toxic response and have a selectivity indexgreater than 5400, which is 10-fold higher than that of AZT.

Example 3.4: VEPEP-3 Mediated Delivery of Proteins in Different CellLines

VEPEP-3 have been used for the delivery of different proteins intodifferent cell lines, including primary cell lines, stem cell lines andchallenging cell lines. Protein uptake was monitored using fluorescencespectroscopy and FACS analysis. GFP/RFP or Fluorescent labelled proteinswere visualized in the different cell lines using fluorescencemicroscopy or FACS sorting (Table 3). In most of the cell lines, theuptake of RFP; GFP, Cy-5 labelled proteins is more than 70% of thecells.

TABLE 3 Efficiency Efficiency Cell lines origin GFP Cy-protein HelaHuman epithelial 67% 90% cervical cancer cells Jurkat Human T lymphocyte80% 90% HepG2 Human hepatocyte 69% 70% C2C12 Mouse myoblast 80% 90% MEFMouse fibroblast 75% 90% HS-68 Human fibroblast 80% 90% CEM-SS Humanmacrophage 80% 80% U2OS Human osteoblast 70% 91% MCF7 Human breast 65%70% adenocarcinoma MT4 Human T lymphocyte 50% 55%

Example 3.5: VEPEP3-Mediated Delivery of Peptide and Protein is notToxic

As shown on FIG. 6, the toxicity of VEPEP-3 particles was investigatedon HeLa and U2OS cells by MTT assay and by monitoring the level ofcyclophilin mRNA measured by Quantigen™ technology (Affymetrix).VEPEP-3a; VEPEP-3b, VEPEP-3c, VEPEP-3d, VEPEP-3e, VEPEP-3f, VEPEP-3g andVEPEP-3h were complexed with short peptide and PNA at ratio 1/20 Notoxicity was detected at levels up to 200 nM, and only a mild toxicitywas observed at the maximum concentration of 1 μM.

Example 3.6: VEPEP-3 Mediated Delivery of PNA Molecule in Different CellLines

VEPEP-3 peptides have been used for the delivery of nucleic acidanalogues (PNA and morpholino) into different cell lines, includingprimary cell lines and challenging cell lines. We demonstrated thatVEPEP-3a and VEPEP-3h form stable complexes with small PNA or morpholinooligonucleotide of 15 mer. We have applied VEPEP-3 strategy for thedelivery of PNA antisense targeting Cyclin B1 as previously described(Morris et al, 2007). Dose-response experiments performed on differentcultured cells revealed that VEPEP-3-mediated delivery of PNA (CyclinB1) induced a robust downregulation higher than 70% of Cyclin B1 proteinlevel (FIG. 7).

Example 3.7: VEPEP-3 Mediated Delivery of Small Hydrophobic Molecules inDifferent Cell Lines

VEPEP-3 peptides have been used for the delivery of different smallfluorescent hydrophobic and charged molecules as well asdoxorubicin/porphyrin/taxol on different cell lines, including primarycell lines and challenging cell lines. VEPEP-3 peptides form stableparticles with small aromatic molecules including doxorubicin orfluorescent dyes. The dissociation constant for small hydrophobicmolecules ranges between 0.01 to 2 μM, depending on the nature of thedyes and of the peptides.

Effect of VEPEP-3a, VEPEP-3c and VEPEP-3g mediated delivery ofdoxorubicin, porphyrin or taxol have been investigated on cancer cellviability. Dose-response experiments performed on cultured cellsrevealed that VEPEP-3 peptide mediated delivery of doxorubicin andporphyrin induced a biological response associated to cell cycle arrestand decrease in viability of MCF-7 and SCK-3-HEK2 cancer cells (FIG. 8shows the results obtained with MCF-7 cells). The impact of carrierpeptides to improve cellular uptake of small molecule drugs wasestimated by following inhibition of proliferation of cancer cells.

IC50 are reported in table 4. IC50 of 0.4 μM and 10 μM were obtained forVEPEP-3/Doxo and free Doxo, respectively. Data demonstrated that Doxo is25 fold more efficient when complexed with VEPEP-3.

TABLE 4 VEPEP-3a Free drug VEPEP-3c VEPEP-3g 1/20 IC50 IC50 1/20 IC501/20 IC50 Drug (μM) (μM) (μM) (μM) Doxo (SKB3) 0.3 10 0.4 0.2 Doxo(MCF7) 0.2 7 0.5 0.2 Porphyrin (MCF7) 0.8 25 1.7 0.5 Porphyrin (SKB3)1.5 17 2.4 1.2 Taxol (MCF7) 0.2 7 0.5 0.7 Taxol (SKB3) 0.5 9 0.9 0.8

Example 4: NANOPEP-3 Formulations and Applications for In Vivo Delivery

NANOPEP particles contain a “peptide-core” or “core shell” correspondingto the association of either VEPEP-3 peptide or any other peptideforming non covalent complexes with its respective cargo, that issurrounded by additional VEPEP-3 “peripheral” peptides stabilizing theparticle and favouring cell membrane association. The efficiency ofNANOPEP is mainly controlled by the size and the charge of theparticles, which should be ranging between 100-200 nm and +5-+20 Volts,respectively. Several combinations can be used for the “core” andperipheral VEPEP-3 can be functionalized or not. The choice of thepeptides in the “core” is dependent on the nature of the cargoes and canbe either VEPEP-6, a peptide of another VEPEP family (VEPEP-9, . . . ),CADY (Crombez et al, 2009a [10]), MPG (Crombez et al, 2009b [11]) orPEP-1 (Chariot: Morris et al, 2001 [8]), etc.

The NANOPEP particles are formed in a two-step process (FIG. 9A): firstthe “core” at molar ratio of 1/5 or 1/10, then the “peripheral” at molarratio of 1/20 up to 1/80. The multilayer organization of the particleallows their oriented functionalization, that will be chosen dependingon the nature of the cellular target/tissue and administration mode.

A three-step protocol (FIG. 9B) has been established when particlefunctionalization takes place via the nitrilotriacetic acid (NTA) linkedto the VEPEP-3. NTA-group is well known as being able to chelate metaland to strongly interact with histidine-tagged proteins. Coating of theparticles with NTA-functionalized VEPEP-3 allows the attachment anyprotein harboring a histidine tag to the particle. This strategy offersthe major advantage of having a common 2 layers particles “NANOPEPHIS”that can be associated to any His-tagged protein. The NANOPEPHISstrategy has been used to coat the particles with specific antibodiestargeting cell surface antigen (EGF, HER-2 or MUC1) or nanobodiesselected by phage display against specific cell lines for targeteddelivery of peptide. NANOPEPHIS-3 strategy can be universally applied toany peptides and proteins harbouring a Histidine cluster in theirsequence.

Example 5: NANOPEP-3 Strategy Applications

NANOPEP-3 strategy has been used for in vivo delivery and targeting ofdifferent cargos and different peptide-based nanoparticles. Differentexamples of NANOPEP-3 applications are reported hereafter.

Example 5.1: NANOPEP-3 Mediated Short Peptide In Vivo Targeted Deliveryafter Systemic Intravenous or Topical Injections

The therapeutic potential of the NANOPEP-3 technology has been validatedin vivo with peptides targeting either CDK2/CYCLIN A/E and CDK4/CYCLIND, essential protein kinases required for the control of cell cycleprogression in G1 and G2 and established therapeutic target in severalcancers. The potency of this technology has been validated in vivo withpeptides targeting interactions between protein kinases and their cyclinregulators, required for entry and progression through mitosis. Theinventors demonstrated that combining peptide C4 or C2 with NANOPEPprevents lung and prostate tumour growth in xenografted mouse models,upon injection every three days of NANOPEP-3/C4 and NANOPEP-3/C2 at 1mg/kg (FIGS. 10A-10B). The “core” shell of the particles was formedusing VEVEP-3a or VEPEP-3f peptide at a molar ratio of 20/1 or 40/1 witha C4 or C2 peptide. VEPEP-3 peptides were solubilised in water and stocksolution of peptide was sonicated for 10 minutes in a water bath beforecomplex formation. Then VEPEP-3/peptide complexes were formed by addingpeptide (C2 or C4) into the peptide solution and incubating at 37° C.for 20-30 minutes to allow the carrier peptide/peptide complexes toform. Then the particles were coated with either VEPEP-3a (NANO-3A) orVEPEP-3f (NANO-3F) peptides depending on the in vivo target. The coatingwas performed by adding the coating peptide at a molar ratio(peptide/peptide) of 1/5 and then incubating for 20 minutes at 37° C.for obtaining stable complexes as shown in FIGS. 9A-9B. The stocksolutions of particles are performed in water and stable for at leastthree weeks at 4° C. Particles can be lyophilized for long time storage;in that case, 5 to 20% of glucose or manitol are added to the particlesolution before lyophylization to stabilize the particles during theprocess. Before administration the particles are diluted inphysiological conditions, in the presence: 0.9% NaCl and 5 to 20%glucose or manitol.

NANOPEP-3/C4 and NANOPEP-3/C2 Delivery Upon Topical and SystemicInjection

The potential of NANOPEP-3 to deliver C2 or C4 peptide in vivo was firstevaluated on human prostate carcinoma cell PC3-xenografted mice (FIGS.10A-10B). The effect of local intratumoral and intravenousadministration of NANOPEP-3/C4 or NANOPEP-3/C2 particles (molar ratio20/1) on the growth of established subcutaneous tumours was evaluated.At day 50, tumor sizes in the control cohort, injected with PBS hadincreased by about 4.1 fold. Upon local intratumoral treatment,reductions of tumor growth by 75% and 80% were observed using 100 μg(0.5 mg/kg) of C4/NANOPEP-3a or 3f and C2/NANOPEP-3a or 3f tumour growthwas completely prevented with 50 μg (1 mg/kg) C2/NANOPEP-3a or 3f &C4/NANOPEP-3a or 3f, respectively (FIGS. 10A-10B). Following systemicintravenous administration, reductions of tumor growth by 45% and 47%were observed using 500 μg (1 mg/kg) of C4/NANOPEP-3 and C2/NANOPEP-3,respectively. In both cases, inhibition of tumour growth was C4 or C2sequence-specific, since scrambled peptide C4S complexed with NANOPEP-3and injected into mice at 2 mg/kg was unable to inhibit tumour growth.The results demonstrated that NANOPEP-3 particles are less efficient viasystemic injection, which is probably due to lower stability in theblood of the particle (see below).

NANOPEP-3 Mediated C2 and C4 Peptide Delivery Upon Systemic Injection

The stability of drug-carrier formulations in vivo and in the bloodcirculation is a major issue for systemic administration oftherapeutics. In order to improve the bioavailability and stability ofthe NANOPEP-3a/peptide particles, these were coated with PEG-VEPEP-3a,thereby rendering them more suitable for systemic administration; thesurface layer of NANOPEP-3a particles was functionalized with aPEG-moiety at the N-terminus of VEPEP-3 (PEG-VEPEP-3a), throughactivation of the N-terminal beta alanine amino group.PEGylated-NANOPEP-3a/C4 particles were obtained stepwise by complexingVEPEP-3 molecules with C4 at a molar ratio of 15/1, followed by coatingof particles with a second layer of PEG-VEPEP3a at ratio 1/10. In orderto analyze if increase in the distribution of C4 peptide associated tofunctionalized-NANOPEP-3a particles directly affects its potency toinhibit tumour growth, the particles were used for systemic intravenousadministration into SKB3-HEK2 xenografted tumor mouse model. 100 μg (0.5mg/kg) of C4 peptide complexed with PEG-NANOPEP-3 at a 1/30 ratio wereinjected intravenously every three days into mice bearing SKB3-HEK2xenografted tumor and a significant reduction in tumor size of 90% wasobserved at day 50 (FIG. 11), which is 10-fold more potent than the nonfunctionalized-NANOPEP-3 nanoparticle, suggesting that PEG-increases thebiodistribution of peptide in the tumour by maintaining peptide in theplasma and by stabilizing the NANOPEP-3 particle.

Example 5.2: NANOPEP-3 Mediated Anti Cyclin B1 PNA Antisens DeliveryUpon Systemic Injection

NANOPEP-3 was used for the delivery of antisense PNA targeting cyclin B1antisense in vivo. NANOPEP-3H/PNA, NANOPEP-3A/PNA free or coated withPEG-VEPEP-3A particles were evaluated directly on the potency to inhibittumour growth; the particles were used for systemic intravenousadministration into SKB3-HEK2 xenografted tumor mouse model. In thelater, the surface layer of NANOPEP-3 particles was functionalized witha PEG-moiety at the N-terminus of VEPEP-3A (PEG-VEPEP-3a), throughactivation of the N-terminal beta alanine amino group.PEGylated-NANOPEP-3/PNA particles were obtained stepwise by complexingVEPEP-3 molecules with PNA at a molar ratio of 10/1, followed by coatingof particles with a second layer of PEG-VEPEP3 at ratio 1/10. 5 μg (0.1mg/kg) and 10 μg of PNA complexed with NANOPEP-3 and PEG-NANOPEP-3 at a1/30 ratio were injected intravenously every three days into micebearing SKB3-HEK2 xenografted tumor. As reported in FIG. 12, at day 50,reductions of tumor growth by 20 and 43% were obtained with 5 μg and 10μg of PNA complexed with NANOPEP-3, respectively. A significantreduction in tumor size of 90% was observed with 5 μg of PNA complexedwith PEG-NANOPEP-3, at day 50 (FIG. 12). Inhibition of tumour growth wasPNA cyclin B1 sequence-specific as 50 μg scrambled PNA complexed withNANOPEP-3 and injected into mice was unable to inhibit tumour growth.These results suggested that VEPEP-3 constitutes a great carrier for invivo delivery of PNA and that PEG-increases the biodistribution of PNAin the tumour by improving the stability of the NANOPEP-3 particle.

Example 5.3: NANOPEP-3-Mediated In Vivo Brain Targeting of Peptide

VEPEP-3 peptides were used to promote brain targeting of peptide-basednanoparticles. VEPEP-3 was used as carrier and coated with VEPEP-3 forbrain targeting. VEPEP-3 peptide was also added as a coating peptide onother peptide-based nanoparticle cargo complexes (including other VEPEPfamily, CADY, PEP, or MPG/peptide “core shell particles”). The cargosused were either a fluorescently-labelled peptide or siRNA. Particleswere formed as reported in FIGS. 9A-9B. In all the cases, 5 μg ofcargoes complexed with peptide carrier were injected intravenously. Thenthe in vivo biodistribution of the cargos was monitored using livefluorescence animal imaging. VEPEP-3/peptide results in a largeaccumulation of Peptide in the brain (FIG. 13), and the presence ofVEPEP-3 coating induced an increase of the cargoes (peptide or siRNA)delivery into the brain. Monitoring fluorescence of labelled cargoesshowed a 5 to 10-fold increase in the brain, in comparison to controlexperiments in the absence of VEPEP-3 coating of the particle. Thepresence of VEPEP-3 coating also increased by a 3-fold factor theknockdown of GAPDH in the brain (FIG. 13). Taken together, these resultsdemonstrated that VEPEP-3 peptide improves significantly brain targetingof peptide-based nanoparticles and can be used for brain targetingeither as a carrier or as a coating layer of already formedpeptide-based nanoparticles.

Example 5.4: NANOPEP-3 Mediated In Vivo Delivery of Cargo Via DifferentAdministration Routes

NANOPEP-3 based particles have been evaluated using differentadministration routes including systemic intravenous, intrarectal,intranasal and transdermal administrations.

A fluorescently labelled peptide or protein (small nanobody) with Alexa700 was complexed into NANOPEP-3 particles. Biodistribution of thefluorescently labelled peptide/protein was evaluated in vivo on Balb6Mouse, 5 hr after a single administration of 10 μg peptide or protein inNANOPEP-3 particles. Intravenous and intrarectal administrations of theNANOPEP-3/peptide or NANOPEP-3/protein complex allowed the delivery ofthe peptide in most of the analyzed tissues, with a significant deliveryin the brain and ganglions (FIGS. 14A-14B). For protein, the deliverywas mainly in lung, liver, kidney, brain and lymphe node. Intranasal andintratracheal administrations allowed the delivery of peptide mainlyinto the brain, lung, liver, pancreas and kidney and protein into thelung, liver and brain. Finally, transdermal administration is limited tothe delivery of the peptide and protein into and through the skin andmuscles, but not in the other tissues (FIGS. 14C-14D).

REFERENCES

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We claim:
 1. A method of delivering a cargo molecule into an individual,comprising administering a nanoparticle or complex into the individual,wherein the nanoparticle or complex comprises a VEPEP-3 cell-penetratingpeptide and said cargo molecule, and wherein the VEPEP-3cell-penetrating peptide comprises an amino acid sequence of: a)X₃X₄X₁X₂X₅X₄X₁X₂X₆X₇X₁X₈X₉X₁₀X₁₁X₁₂X₁₃ (SEQ ID No: 11), wherein X₁ inpositions 3, 7, and 11 are, independently from each other, F or W, X₂ inpositions 4 and 8 are, independently from each other, F, W or Y, X₃ isbeta-A or S, X₄ in positions 2 and 6 are, independently from each other,K, R or L, X₅ is E, R or S, X₆ is R, T or S, X₇ is E, R or S, X₈ isnone, F or W, X₉ is P or R, X₁₀ is R or L, X₁₁ is K, W or R, X₁₂ is R orF and X₁₃ is R or K; b) X₃KX₁₄WWERWWRX₁₄WPRKRK (SEQ ID No: 9), whereinX₃ is a beta-alanine or a serine, X₁₄ in positions 3 and 11 are,independently from each other, a non-natural amino acid, and there is ahydrocarbon linkage between the two non-natural amino acids; or c)X₃RWWX₁₄LWWRSWX₁₄RLWRR (SEQ ID No: 10), wherein X₃ is a beta-alanine ora serine, X₁₄ in positions 5 and 12 are, independently from each other,a non-natural amino acid, and there is a hydrocarbon linkage between thetwo non-natural amino acids.
 2. The method of claim 1, wherein thenanoparticle or complex comprises a core comprising the VEPEP-3cell-penetrating peptide and the cargo, wherein the cargo molecule iscomplexed to the VEPEP-3 cell-penetrating peptide.
 3. The method ofclaim 1, wherein the VEPEP-3 cell-penetrating peptide further comprisesa) an acetyl group or a poly-ethylene glycol covalently linked to theN-terminal end of the amino acid sequence, and/or b) a cysteamide groupcovalently linked to the C-terminal end of the amino acid sequence. 4.The method of claim 1, wherein the VEPEP-3 cell-penetrating peptidecomprises an amino acid sequence X₃X₄WX₂EX₄WX₂X₆X₇X₁PRX₁₁RX₁₃ (SEQ IDNo: 12), wherein X₁ is F or W, X₂ in positions 4 and 8 are,independently from each other, F, W or Y, X₃ is beta-A or S, X₄ inpositions 2 and 6 are, independently from each other, K or R, X₆ is T orR, X₇ is E or R, X₁₁ is R or K, and X₁₃ is R or K.
 5. The method ofclaim 4, wherein the VEPEP-3 cell-penetrating peptide is selected fromthe group consisting of: (SEQ ID No: 1) X₃KWFERWFREWPRKRR, (SEQ ID No:2) X₃KWWERWWREWPRKRK, (SEQ ID No: 3) X₃RWWEKWWTRWPRKRK, and (SEQ ID No:4) X₃RWYEKWYTEFPRRRR,

wherein X₃ is beta-A or S.
 6. The method of claim 1, where in theVEPEP-3 cell-penetrating peptide comprises an amino acid sequence:X₃X₄X₁WX₅X₄X₁WX₆X₇WX₈X₉X₁₀WX₁₂R (SEQ ID No: 13), wherein X₁ in positions3 and 7 are, independently from each other, F or W, X₃ is beta-A or S,X₄ in position 2 is K, R or L, X₄ in position 6 is L or R, X₅ is R or S,X₆ is R or S, X₇ is R or S, X₈ is F or W, X₉ is R or P, X₁₀ is L or R,and X₁₂ is R or F.
 7. The method of claim 6, wherein cell-penetratingpeptide is selected from the group consisting of: (SEQ ID No: 5)X₃RWWRLWWRSWFRLWRR, (SEQ ID No: 6) X₃LWWRRWWSRWWPRWRR, (SEQ ID No: 7)X₃LWWSRWWRSWFRLWFR, and (SEQ ID No: 8) X₃KFWSRFWRSWFRLWRR,

wherein X₃ is beta-A or S.
 8. The method of claim 1, wherein thenanoparticle or complex is nanoparticle.
 9. The method of claim 8,wherein the size of the nanoparticle is about 20 to about 300 nm. 10.The method of claim 8, wherein the nanoparticle further comprises alayer of peripheral cell-penetrating peptides, wherein the core iscoated with the layer of peripheral cell-penetrating peptides.
 11. Themethod of claim 10, wherein the peripheral cell-penetrating peptidescomprises an amino acid sequence of: a)X₃X₄X₁X₂X₅X₄X₁X₂X₆X₇X₁X₈X₉X₁₀X₁₁X₁₂X₁₃ (SEQ ID No: 11), wherein X₁ inpositions 3, 7, and 11 are, independently from each other, F or W, X₂ inpositions 4 and 8 are, independently from each other, F, W or Y, X₃ isbeta-A or S, X₄ in positions 2 and 6 are, independently from each other,K, R or L, X₅ is E, R or S, X₆ is R, T or S, X₇ is E, R or S, X₈ isnone, F or W, X₉ is P or R, X₁₀ is R or L, X₁₁ is K, W or R, X₁₂ is R orF and X₁₃ is R or K; b) X₃KX₁₄WWERWWRX₁₄WPRKRK (SEQ ID No: 9), whereinX₃ is a beta-alanine or a serine, X₁₄ in positions 3 and 11 are,independently from each other, a non-natural amino acid, and there is ahydrocarbon linkage between the two non-natural amino acids; or c)X₃RWWX₁₄LWWRSWX₁₄RLWRR (SEQ ID No: 10), wherein X₃ is a beta-alanine ora serine, X₁₄ in positions 5 and 12 are, independently from each other,a non-natural amino acid, and there is a hydrocarbon linkage between thetwo non-natural amino acids.
 12. The method of claim 1, wherein thecargo molecule is selected from the group consisting of peptides,proteins, peptide analogs, uncharged oligonucleotides, PNAs and smallhydrophobic molecules.
 13. The method of claim 12, wherein the cargomolecule is a protein.
 14. The method of claim 1, wherein thenanoparticle or complex is administered intravenously, intratumorally,topically, intrarectally, intranasally, transdermally, or intradermally,via a mouth spray, or subcutaneously.
 15. The method of claim 1, whereinthe individual is a human.
 16. The method of claim 1, wherein theindividual has a cancer.
 17. The method of claim 1, wherein theindividual has a brain disease or lymph node disease, and wherein theVEPEP-3 cell-penetrating peptide comprises an amino acid sequence of: a)X₃KX₁₄WWERWWRX₁₄WPRKRK (SEQ ID No: 9), wherein X₃ is a beta-alanine or aserine, X₁₄ in positions 3 and 11 are, independently from each other, anon-natural amino acid, and there is a hydrocarbon linkage between thetwo non-natural amino acids; or b) X₃RWWX₁₄LWWRSWX₁₄RLWRR (SEQ ID No:10), wherein X₃ is a beta-alanine or a serine, X₁₄ in positions 5 and 12are, independently from each other, a non-natural amino acid, and thereis a hydrocarbon linkage between the two non-natural amino acids. 18.The method of claim 1, wherein the nanoparticle or complex is ananoparticle comprising a core coated by the VEPEP-3 cell-penetratingpeptide, wherein the core comprises the cargo molecule, and wherein thecargo molecule is complexed to a first entity selected from the groupconsisting of cell-penetrating peptides, liposomes, polycationicstructures and carbon nanoparticles.
 19. The method of claim 19, whereinthe first entity is a cell-penetrating peptide selected from the groupconsisting of VEPEP-6a (SEQ ID No: 29), VEPEP-6b (SEQ ID No: 30),VEPEP-6c (SEQ ID No: 31), VEPEP-6d (SEQ ID No: 32), VEPEP-6e (SEQ ID No:33), VEPEP-6f (SEQ ID No: 34), VEPEP-9a1 (SEQ ID No: 35), VEPEP-9a2 (SEQID No: 36), VEPEP-9b1 (SEQ ID No: 37), VEPEP-9b2 (SEQ ID No: 38),VEPEP-9c1 (SEQ ID No: 39), VEPEP-9c2 (SEQ ID No: 40), CADY, MPG, PEP-1,PPTG 1, and poly Arginine.
 20. A method of treating a disease orcondition in an individual, comprising administering to the individualan effective amount of a complex or nanoparticle comprising a VEPEP-3cell-penetrating peptide and a cargo molecule selected from the groupconsisting of peptides, proteins, peptide analogs, unchargedoligonucleotides, PNAs and small hydrophobic molecules, wherein theVEPEP-3 cell-penetrating peptide comprises an amino acid sequenceselected from: a) X₃X₄X₁X₂X₅X₄X₁X₂X₆X₇X₁X₈X₉X₁₀X₁₁X₁₂X₁₃ (SEQ ID No:11), wherein X₁ in positions 3, 7, and 11 are, independently from eachother, F or W, X₂ in positions 4 and 8 are, independently from eachother, F, W or Y, X₃ is beta-A or S, X₄ in positions 2 and 6 are,independently from each other, K, R or L, X₅ is E, R or S, X₆ is R, T orS, X₇ is E, R or S, X₈ is none, F or W, X₉ is P or R, X₁₀ is R or L, X₁₁is K, W or R, X₁₂ is R or F and X₁₃ is R or K; b) X₃KX₁₄WWERWWRX₁₄WPRKRK(SEQ ID No: 9), wherein X₃ is a beta-alanine or a serine, X₁₄ inpositions 3 and 11 are, independently from each other, a non-naturalamino acid, and there is a hydrocarbon linkage between the twonon-natural amino acids; and c) X₃RWWX₁₄LWWRSWX₁₄RLWRR (SEQ ID No: 10),wherein X₃ is a beta-alanine or a serine, X₁₄ in positions 5 and 12 are,independently from each other, a non-natural amino acid, and there is ahydrocarbon linkage between the two non-natural amino acids.
 21. Themethod of claim 20, wherein the disease or condition is a cancer. 22.The method of claim 20, wherein the disease or condition is a braindisease or lymph node disease, and wherein the VEPEP-3 cell-penetratingpeptide comprises an amino acid sequence of: a) X₃KX₁₄WWERWWRX₁₄WPRKRK(SEQ ID No: 9), wherein X₃ is a beta-alanine or a serine, X₁₄ inpositions 3 and 11 are, independently from each other, a non-naturalamino acid, and there is a hydrocarbon linkage between the twonon-natural amino acids; or b) X₃RWWX₁₄LWWRSWX₁₄RLWRR (SEQ ID No: 10),wherein X₃ is a beta-alanine or a serine, X₁₄ in positions 5 and 12 are,independently from each other, a non-natural amino acid, and there is ahydrocarbon linkage between the two non-natural amino acids.